The present invention relates to novel pyrrolyltetrahydroquinoxalinediones, processes for the preparation thereof and the use thereof for controlling diseases.
Excitatory amino acids, in particular glutamic acid, are wide-spread in the central nervous system. The excitatory amino acid glutamate acts as transmitter substance for receptors of which various subtypes are known. One subtype is called, for example, the NMDA receptor after the specific agonist N-methyl-D-aspartate. This NMDA receptor has various binding sites for agonists and antagonists. The amino acid glycine likewise binds to the NMDA receptor and modulates the effect of the natural agonist glutamic acid. Antagonists on this glycine binding site may accordingly show antagonistic effects on the NMDA receptor and inhibit an overexcitation of this receptor.
Two other subtypes of glutamate receptors are the AMPA receptor and the kainate receptor which are each called after the specific agonists 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainic acid. In a similar way to the NMDA receptor already mentioned, antagonists of these receptors can likewise inhibit overexcitation.
Elevated glutamate activity occurs in a number of neurological disorders or psychological disturbances and leads to states of overexcitation or toxic effects in the CNS.
Derivatives of quinoxaline-2,3(1H,4H)-dione II 
have been described as glutamate antagonists in several publications, such as EP-A 374 534 and EP-A 260 467. Many of the known derivatives are unsubstituted in the heterocyclic quinoxaline fragment (II, R1, R2=hydrogen). Furthermore, derivatives where R1 in II is a radical which is not hydrogen are also known. Thus, EP-A 377 112 and EP-A 374 534 have claimed N-hydroxyquinoxalines (II; R1=OR4). EP-A 315 959, DE-A 4 135 871, WO 91/13 878 and WO 92/07 847 describe alkyl radicals as R1 in II, and the alkyl chain can also be substituted by acids, esters or amides. Alkyl acids (=R1) are likewise mentioned in J. R. Epperson et al. Bioorg. and Med. Chemistry Lett. 3 (1993), 2801-4.
Quinoxalinedione derivatives II which carry a heterocycle as substituent R3 are likewise known. Thus, EP-A 556 393 describes imidazoles, triazoles and pyrazoles. Quinoxaline dione derivatives which carry a pyrrolyl radical as R3 have been described as glutamate antagonists in EP-A 572 852.
The substances according to the invention are suitable for treating all disorders in which a beneficial effect can be expected from glutamate antagonists.
Suitable indications are neurotoxic disturbances, especially acute and chronic oxygen/(nutrient) deficiency/states of the central nervous system. By these are meant acute hypoxic or ischemic states which occur, for example, as a consequence of cerebral infarct, subarachnoid hemorrhage or vasospasms of other etiology, also following heart/circulatory failure, eg. in cardiac arrest, cardiac arrhythmias or circulatory collapse; CNS damage following hypoglycemia, as a consequence of perinatal asphyxia or after craniocerebral trauma, spinal cord trauma, transient ischemic attacks (TIAs), prolonged reversible ischemic neurological deficits (PRINDs) and multi-infarct dementia and atherosclerotic dementia, and migraine.
Other possible indications are neurodegenerative disorders, eg. Parkinson""s disease, Huntington""s chorea, Alzheimer""s disease, amyotropic lateral sclerosis (ALS).
Furthermore, glutamate antagonists may be suitable for use as antiepileptics, as anxiolytics and as antidepressants, and for the treatment of pain, likewise for the treatment is schizophrenia, of withdrawal symptoms in addicts, and as muscle relaxants in cases of spasticity of the skeletal muscles, eg. in multiple sclerosis (MS).
The invention relates to novel pyrrolylquinoxalinediones of the formula I 
and their tautomeric and isomeric forms, and their physiologically tolerated salts, where the variables have the following meanings:
R1 
hydrogen, cyclohexyl
an aliphatic radical which has 1 to 4 carbon atoms and can carry the radical xe2x80x94COOR5, where R5 is hydrogen or C1-C4-alkyl, preferably xe2x80x94(CH2)mxe2x80x94COOR5 with m=1-4, particularly preferably xe2x80x94CH2COOH,
R2
COOH, COOxe2x80x94C1-C4, alkyl, xe2x80x94COOxe2x80x94(CH2)mxe2x80x94Ph, xe2x80x94CONR6R7 and

xe2x80x83where m can be an integer from 1 to 6, R6 can be hydrogen, C1-C4-alkyl or OH and R7 can be hydrogen, C1-C4-alkyl, 
xe2x80x83where all the phenyl or pyridyl rings present in R2 can also be substituted by up to 3 of the following radicals:
C1-C4-alkyl, halogen, xe2x80x94Oxe2x80x94C1-C4-alkyl, xe2x80x94OCF3, NO2, CN, xe2x80x94COOR5 or xe2x80x94CONHR5,
R3
CF3, NO2, CN, and
R4 
hydrogen and
R3 and R4 
can together be a fused-on benzene ring.
Preferred compounds according to the invention are quinoxaline-dione derivatives of the formula 
where
R2
is xe2x80x94COOH or xe2x80x94CONHR7 where R7 is hydrogen, xe2x80x94CH2-C6H5,

R3 
is NO2 or CF3 and R4 is hydrogen, or
R3 and R4 are a fused-on benzene ring.
Very particularly preferred compounds are:
1-Carboxymethyl-7-(3-carboxy-1-pyrrolyl)-6-nitroquinoxaline-2,3(1H, 4H)-dione,
1-Carboxymethyl-7-(3-carboxy-1-pyrrolyl)-6-trifluoromethyl-quinoxaline-2,3-(1H, 4H)-dione,
1-Carboxymethyl-7-(3-carboxy-1-pyrrolyl)-benzo[f]quinoxaline-2,3-(1H, 4H)-dione,
7-(3-Benzylcarbamoyl-1-pytrolyl)-1-carboxymethyl-6-trifluoro-methylquinoxaline-2,3-(1H, 4H)-dione,
7-(3-Benzylcarbamoyl-1-pyrrolyl)-1-carboxymethyl-6-nitro-quinoxaline-2,3-(1H, 4H)-dione,
1-Carboxymethyl-6-nitro-7-(3-(3-phenylpropylcarbamoyl)-1-pyrrolyl)-quinoxalin-2,3(1H,4H)-dione,
1-Carboxymethyl-6-nitro-7-(3-(4-pyridinylmethylcarbamoyl)-1-pyrrolyl)quinoxaline-2,3(1H, 4H)-dione,
7-(3-Carbamoyl-1-pyrrolyl)-1-carboxymethyl-6-nitroquinoxaline-2,3(1H,4H)-dione,
1-Carboxymethyl-6-nitro-7-(3-(4-nitrobenzylcarbamoyl)-1-pyrrolyl)quinoxaline-2,3(1H,4H)-dione.
It has been found, surprisingly, that the present pyrrole-carboxylic acid derivatives have advantages by comparison with the compounds mentioned in EP-A 572 852.
The compounds I according to the invention can be prepared in the way outlined in reaction scheme 1 below.
Synthesis of the aldehydes III has been described in EP-A 572 852. These aldehydes can be oxidized to the carboxylic acids I a according to the invention by customary literature methods which are listed, for example, in R. C. Larock, Comprehensive Organic Transformations, 1989, VCH Publisher, page 838 et seq. Particularly used for example is potassium permanganate in solvents such as acetone at 25-60xc2x0 C. If there is an ester residue in R1, this can subsequently be hydrolyzed with acids and bases to result in the dicarboxylic acid I d. The hydrolysis is preferably carried out with lithium hydroxide in tetrahydrofuran/water mixtures at room temperature. 
The pyrrolecarboxylic acid I a can be converted by reaction with amines or alcohols into the derivatives I b. In this case, the acid group COOH is activated in a suitable manner to COL where L is a leaving group such as azide, imidazole and others which are listed in R. C. Larock, Comprehensive Organic Transformations, New York 1989, pages 972 et seq. Subsequent addition of the reactants HNR6R7 or alcohols results in the products I b according to the invention. If the radical R1 has an ester group, this can be subjected to hydrolysis similar to the above with acids and bases to give the carboxylic acid, resulting in the derivatives I c according to the invention.
The compounds according to the invention are antagonists of the excitatory amino acid glutamate, in particular antagonists of the glycine binding site of the NMDA receptor, of the AMPA receptor and of the kainate receptor.
The pharmacological activity of the compounds I was investigated on isolated membrane material from rat cerebra. For this purpose, the membrane material was treated in the presence of the compounds according to the invention with the radiolabeled substance 3H-2-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (3H-AMPA), [3H]-glycine or [3H]-kainate which bind to specific receptors (AMPA, NMDA or kainate receptors). The radioactivity of the treated membranes was then measured by scintillation counting. It was possible to determine from the bound radioactivity the amounts of bound 3H-AMPA, [3H]-glycine or [3H]-kainate, or in each case the displaced amounts of these radiolabeled substances.
The dissociation constant KI (I=inhibitor) which emerges from this and is a measure of the displacing action of the agent according to the invention was found by iterative nonlinear regression analysis using the statistical analysis system (SAS) on an IBM computer, similar to the xe2x80x9cLigandxe2x80x9d program of P. J. Munson and D. Rodbard (Analytical Biochem. 107 (1980) 220, Ligand: Versatile Computerized Approach for Characterization of Ligand Binding Systems).
The following in vitro investigations were carried out:
1. Binding of 3H-2-amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (3H-AMPA)
To prepare the membrane material, freshly removed rat cerebra were homogenized together with 15 times the volume of a buffer solution A composed of 30 mM tris(hydroxymethyl)methylamine hydrochloride (TRIS-HCl) and 0.5 mM ethylenediaminetetraacetic acid (EDTA), pH 7.4, using an Ultra-TurraxR. The suspension was centrifuged at 48000 g for 20 min. After removal of the supernatant liquid, the protein-containing membrane material in the sediment was washed three times by suspension in buffer solution A and subsequent centrifugation at 48000 g for 20 minutes each time. The membrane material was then suspended in 15 times the volume of buffer solution A and incubated at 37xc2x0 C. for 30 min. The protein material was subsequently washed twice by centrifugation and suspension and stored at xe2x88x9270xc2x0 C. until used. For the binding assay, the protein material was thawed at 37xc2x0 C. and washed twice by centrifugation at 48000 g (20 min) and subsequent suspension in a buffer solution B composed of 50 mM TRIS-HCl, 0.1 M potassium thiocyanate and 2.5 mM calcium chloride, pH 7.1. Subsequently, 0.25 mg of membrane material, 0.1 xcexcCi of 3H-AMPA (60 Ci/mmol) and compound I were dissolved in 1 ml of buffer solution B and incubated on ice for 60 min. The incubated solution was filtered through a CF/B filter (from Whatman) which had previously been treated with a 0.5% strength aqueous solution of polyethyleneimine for at least 2 hours. The membrane residue was subsequently washed with 5 ml of cold buffer solution B in order to separate bound and free 3H-AMPA from one another. After measurement of the radioactivity of the bound 3H-AMPA in the membrane material by scintillation counting, the KI was determined by regression analysis of the displacement plots.
A kI of  less than 10 xcexcM was found for 7-(3-carboxy-1-pyrrolyl)-1-(ethoxy-carbonylmethyl)-6-nitroquinoxaline-2,3(1H,4H)-dione (Example 2). The substance is accordingly distinctly more active than the very closely related aldehyde 1-(ethoxycarbonylmethyl)-7-(3-formyl-1-pyrrolyl)-6-nitroquinoxaline-2,3(1H,4H)-dione (Example 80 in EP 572 852).
2. Binding of [3H]-glycine
To prepare the membranes for the 3H-glycine binding assay, freshly removed rat hippocampi were homogenized in 10 times the volume of preparation buffer (50 mM tris-HCl, 10 mM EDTA) using a Potter homogenizer. The homogenate was centrifuged at 48000xc3x97g for 20 min. The supernatant was discarded, and the membranes present in the pellet were washed 2xc3x97 by resuspension and centrifugation at 48000xc3x97g (20 min each time). The resuspended membranes were frozen in liquid nitrogen and thawed again at 37xc2x0 C. After another washing step, the membrane suspension was incubated in a shaking water bath at 37xc2x0 C. for 15 min. After a further 4 washing steps (centrifugation at 48000xc3x97g for 20 minutes each time and resuspension in preparation buffer), the membranes were stored at xe2x88x9270xc2x0 C. until used further.
The frozen membranes were thawed at 37xc2x0 C. and washed 2xc3x97 by centrifugation at 48000xc3x97g (20 min) and subsequent resuspension in binding buffer (50 mM tris-HCl pH 7.4, 10 mM MgCl2). An incubation mixture contained 0.25 mg of protein (membranes), 25 nM 3H-glycine (16 Ci/mmol) and the substances to be tested in a total of 0.5 ml of binding buffer. The nonspecific binding was determined by adding 1 mM glycine.
After incubation at 4xc2x0 C. for 60 min, bound and free ligand were separated from one another by filtration through GF/B filters and subsequent washing with about 5 ml of ice-cold binding buffer.
The radioactivity remaining on the filters is determined by liquid scintillation counting. The dissociation constants were calculated from the displacement plots using an iterative nonlinear fitting program or in accordance with the equation of Cheng and Prusoff.
3. Binding of [3H]-kainate
To prepare the membranes for the [3H]-kainate binding assay, freshly removed rat cerebra were homogenized in 15 times the volume of preparation buffer (30 mM tris-HCl pH 7.4, 0.5 mM EDTA) using an Ultra-TurraxR. The homogenate was centrifuged at 48000xc3x97g for 20 min. The supernatant was discarded, and the membranes present in the pellet were washed a total of 3xc3x97 by resuspension in preparation buffer and centrifugation at 48000xc3x97g (20 min each time). After the third washing step, the membranes were washed 2xc3x97 by centrifugation and resuspension and stored at xe2x88x9270xc2x0 C. until used further.
The frozen membranes were thawed at 37xc2x0 C., suspended in binding buffer (50 mM tris-HCl pH 7.4) and centrifuged at 48000xc3x97g for 20 min. The membranes present in the pellet were resuspended in binding buffer. An incubation mixture contained 0.25 mg of protein (membranes), 0.058 xcexcCi (58 Ci/mmol) of 3H-kainate and the substances to be tested in a total of 1 ml of binding buffer. The nonspecific binding was determined in the presence of 0.1 mM glutamate. After incubation on ice for 60 minutes, bound and free ligand were separated from one another by filtration through CF/B filters and subsequent washing with 5 ml of ice-cold binding buffer. The CF/B filters had previously been treated with 0.5% polyethyleneimine for at least 2 h. The displacement plots were analyzed, and the dissociation constants were calculated using a nonlinear fitting program or in accordance with the equation of Cheng and Prusoff.
To demonstrate the in vivo activity of the novel substances, results from the following test arrangements can be used:
Anticonvulsant Effect (Maximum electric shock in mice)
Tonic spasms of the rear limbs are induced in mice by a maximum electric shock. The occurrence of spasms can be antagonized by pretreatment with test substances. This antispasmodic effect is evidence of the possible use of a substance as antiepileptic.
Protection Against Cerebral Overexcitation by Excitatory Amino Acids (NMDA or AMPA antagonism in vivo, mouse)
Intracerebral administration of excitatory amino acids (=EAA) induces such massive overexcitation that this leads to spasms and to the death of the animals within a short time. These signs can be inhibited by systemic, eg. intraperitoneal, administration of centrally acting EAA antagonists. Since excessive activation of EAA receptors in the central nervous system plays a significant part in the pathogenesis of various neurological disorders, it is possible to conclude from the detected EAA antagonism in vivo that the substances can be used for the therapy of such CNS disorders. These include, inter alia, focal and global ischemias, trauma, epilepsy and various neurodegenerative disorders such as Huntington""s chorea, Parkinson""s disease etc.
The compounds I according to the invention are suitable as drugs for human and veterinary medicine and can be used to produce drugs for the treatment of neurodegenerative disorders and neurotoxic disturbances of the central nervous system and for producing antiepileptics, anxiolytics, antidepressants and antinociceptives.
The drug preparations according to the invention contain a therapeutically effective amount of the compounds I in addition to conventional pharmaceutical ancillary substances.
For local external use, eg. in dusting powders and ointments, the agents can be present in the usual concentrations. The agents are, as a rule, present in an amount of from 0.0001 to 1% by weight, preferably 0.001 to 0.1% by weight.
For internal use, the preparations are administered in single doses. 0.1 to 100 mg are given per kg of body weight in a single dose. The preparations can be administered in one or more doses each day, depending on the nature and severity of the disorders.
Appropriate for the required mode of administration, the drug preparations according to the invention contain conventional excipients and diluents in addition to the agent. Pharmaceutical ancillary substances possible for local external use are, for example, ethanol, isopropanol, ethoxylated castor oil, ethoxylated hydrogenated castor oil, polyacrylic acid, polyethylene glycol, polyethylene glycol stearate, ethoxylated fatty alcohols, liquid paraffin, petrolatum and lanolin. Examples suitable for internal use are lactose, propylene glycol, ethanol, starch, talc and polyvinylpyrrolidone.
It is furthermore possible for antioxidants such as tocopherol and butylated hydroxyanisole and butylated hydroxytoluene, flavor-improving additives, stabilizers, emulsifiers and lubricants to be present.
The substances which are present in the preparation in addition to the agent, and the substances used in the production of the pharmaceutical preparation, are toxicologically acceptable and compatible with the agent in each case. The drug preparations are produced in a conventional way, for example by mixing the agent with the conventional excipients and diluents.
The drug preparations can be administered in various ways such as orally, parenterally, subcutaneously, intraperitoneally and topically. Thus, possible presentations are tablets, emulsions, infusion and injection solutions, pastes, ointments, gels, creams, lotions, dusting powders and sprays.